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Enhancing endonuclease based gene editing in primary cells — Seattle Children's Hospital (dba Seattle Children's Research Institute) (US20200377911A1)

ABSTRACT

Abstract

Disclosed herein are nuclease-based systems for genome editing and methods of using the system for genome editing. Also, disclosed are approaches to enhance Cas9-mediated gene editing efficiency in primary human cells with minimal toxicity when using adeno-associated virus vectors (AAV) to express the guide RNAs necessary for CRISPR/Cas9-based genome editing in the presence of helper proteins.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/152,960 filed on May 12, 2016 which claims the benefit of U.S. Prov. App. No. 62/161,104 filed on May 13, 2015, which are each hereby expressly incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SCRI094D1SEQLIST created on Jan. 10, 2020 and which is approximately 70 kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

Aspects of the disclosure provided herein are generally related to endonuclease-based gene editing systems and methods. Some aspects of the disclosure provided herein are related to the CRISPR/Cas9 gene editing system.

BACKGROUND

Endonuclease-based systems have rapidly become significant gene editing tools in biomedical research, with their application for gene disruption and/or gene targeting demonstrated in a variety of cultured cell and model organism systems.

Endonuclease-based systems for gene editing allow scientists to edit genomes with unprecedented precision, efficiency, and flexibility. Examples of endonuclease-based approaches for gene editing include systems comprising, without limitations, zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases (such as MegaTALs), and CRISPR/Cas9.

SUMMARY

The present disclosure provides several methods for applying CRISPR/Cas9 in primary cells in which an mRNA is used to express Cas9, and simultaneously, mRNA is used to transiently express two adenoviral proteins, E40RF6 and an H373A or H354 mutant version of E1B55K. The wild type E40RF6 and EB55K proteins relieve post-entry defects for expression from AAV vectors; however, if the wild type EB55K or E40RF6 proteins are used, they disable an important protein complex involved in DNA repair (known as the MRN complex), which leads to cell cycle arrest and high toxicity due to lack of repair of DNA breaks. Instead of using the wild type proteins mutants of E1B55K, which do not disable the MRN complex are utilized. Co-expression of Cas9 with E40RF6/E1B55K mutants results in sufficient relief of the post-entry restriction on AAV expression while maintaining intact DNA repair. This allows for a substantial improvement in Cas9-mediated gene editing efficiency with minimal toxicity when an AAV vector is used to express the guide RNA's necessary for Cas9 targeting.

Some alternatives of the system provided herein, comprise endonucleases so as to provide additional tools useful in gene disruption. Several alternatives, for example, relate to systems utilizing CRISPR/Cas9 systems and methods for enhancing the efficiency of inactivation of a target gene concurrently with endonucleases. More alternatives relate to the inactivation of a target gene for therapeutic, agricultural and/or other commercially useful purposes utilizing one or more of the systems described herein. Still more alternatives relate to the production of autologous and/or non-autologous primary cells having an inactivated target gene and the use of these cells for therapeutic and/or other commercial applications.

In some alternatives, a system for editing at least one target gene in a cell is provided, the system comprising a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector; a second nucleic acid sequence encoding a Cas9 protein, a derivative or fragment thereof; a third nucleic acid sequence encoding a first adenoviral protein; and a fourth nucleic acid sequence encoding a second adenoviral protein. In some alternatives of the system, the cell is a eukaryotic cell. In some alternatives of the system, the cell is a mammalian cell. In some alternatives of the system, the cell is a human cell. In some alternatives of the system, the cell is a primary cell. In some alternatives of the system, the cell is not a transformed cell. In some alternatives of the system, the cell is a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell or an intestinal cell.

In some alternatives of the system, the vector is a viral vector. In some alternatives of the system, the viral vector is an Adeno-associated virus (AAV) vector. In some alternatives of the system, the second nucleic acid encoding the Cas9 protein, a derivative or fragment thereof is an mRNA. In some alternatives of the system, the second nucleic acid sequence encoding the Cas9 protein is codon optimized for expression in a eukaryotic cell, such as a human cell. In some alternatives of the system, the Cas9 protein, a derivative or fragment thereof is from S. pyogenes . In some alternatives of the system, the third nucleic acid encoding the first adenoviral protein is an mRNA. In some alternatives of the system, the mRNA is codon optimized for expression in a eukaryotic cell, such as a human cell. In some alternatives of the system, the first adenoviral protein is E40RF6. In some alternatives of the system, the fourth nucleic acid encoding the second adenoviral protein is an mRNA. In some alternatives of the system, the fourth nucleic acid encoding the second adenoviral protein is codon optimized for expression in a eukaryotic cell, such as a human cell. In some alternatives of the system, the second adenoviral protein is an E1B55K mutant. In some alternatives of the system, the first, second, third and fourth nucleic acid sequences are joined to regulatory elements that are operable in a eukaryotic cell, such as a human cell. In some alternatives of the system, the first nucleic acid sequence encoding the CRISPR guide RNA is operably linked to a regulatory element. In some alternatives of the system, the nucleic acid sequence encoding the CRISPR guide RNA is operably linked to a promoter, for example, a U6 promoter. In some alternatives of the system, the first nucleic acid sequence encoding the CRISPR guide RNA is constitutively expressed.

In some alternatives, a method of editing at least one target gene in a cell is provided, the method comprising introducing into a cell a first vector that comprises a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in said cell; introducing into said cell a second nucleic acid sequence encoding a Cas9 protein, a derivative or fragment thereof, introducing into said cell a third nucleic acid sequence encoding a first adenoviral protein; and introducing into said cell a fourth nucleic acid sequence encoding a second adenoviral protein. In some alternatives of the method, the cell is a eukaroytic cell. In some alternatives of the method, the cell is a mammalian cell. In some alternatives of the method, the cell is a human cell. In some alternatives of the method, the cell is a primary cell. In some alternatives of the method, the cell is not a transformed cell. In some alternatives of the method, the cell is a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell or an intestinal cell.

In some alternatives of the method, the first vector comprising the first nucleic acid sequence encoding the CRISPR guide RNA is a viral vector. In some alternatives of the method, the viral vector is an Adeno-associated virus (AAV) vector. In some alternatives of the method, the second, third and fourth nucleic acid sequences are mRNA. In some alternatives of the method, the mRNAs are codon optimized for expression in a eukaryotic cell, such as a human. In some alternatives of the method, the Cas9 protein, a derivative or fragment thereof is from S. pyogenes . In some alternatives of the method, the first adenoviral protein is E40RF6. In some alternatives of the method, the second adenoviral protein is a EB55K mutant. In some alternatives of the method, the CRISPR guide RNA is complimentary to a target gene of interest. In some alternatives of the method, the CRISPR guide RNA is complimentary to a target gene of interest. In some alternatives of the method, the first, second, third and fourth nucleic acid sequences are transiently introduced into the cell. In some alternatives of the method, the first, second, third and fourth nucleic acid sequences are not permanently introduced into the cell. In some alternatives of the method, the introducing of the first, second, third and fourth nucleic acid sequences into the cell does not transform the cell. In some alternatives of the method, the target gene is a selected or identified gene of interest. In some alternatives of the method and/or the system, the second, third, or fourth nucleic acid sequence is provided on a vector. In some alternatives, a method of editing at least one target gene in a cell is practiced by introducing into a cell any of the alternatives of the system described herein.

In some alternatives, a method of treating, ameliorating, or inhibiting a disease and/or a condition in a subject is practiced by providing to a subject having a disease and/or a condition any of the alternatives of the system described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of mRNA and AAV vector constructs used for TCR locus targeting.

FIG. 1B shows T7 assay of editing-induced insertions-deletions (indels) at TCRα locus.

FIG. 1C shows the effect of Cas9-T2A-mCherry mRNA dose on expression at 24 h post transfection.

FIG. 1D shows the effect of increasing AAV dose on TCRα knockout.

FIG. 1E shows flow cytometry data related to comparison of TCRα knockout with single stranded (ss) versus self-complementary (sc) AAV for guide expression in T-cells.

FIG. 2A shows data related to relief of post-entry restriction of AAV-mediated gene expression.

FIG. 2B shows data related to comparison of effect of E40RF6/E1B55K proteins on self-complementary and single-stranded AAV6-mediated gene expression

FIG. 2C shows data related to MRN inactivation and its effect of relief of post-entry restriction on AAV-mediated expression.

FIG. 3A shows the effect of E40RF6/E1B55K mutants on AAV-driven GFP expression.

FIG. 3B shows the effect of E40RF6/E1B55K-H373 expression on AAV transduction.

FIG. 3C shows a comparison of E1B55K, E40RF6 mutants effects on AAV transduction.

FIG. 3D shows a comparison of E1B55K, E40RF6 mutants effects on AAV-driven GFP expression.

FIG. 4A - FIG. 4C show data related to CRISPR-mediated gene knockout in primary human T-cells through use of adenoviral E40RF6/E1B55K proteins.

FIG. 5A - FIG. 5C show data related to the effect of E40RF6/E1B55K MRN mutants on indel spectra.

FIG. 6 shows the effect of E40RF6/E1B55K proteins on non-homologous AAV insertion.

FIG. 7A - FIG. 7B show data related to implementation of CRISPR/Cas9 with mRNA/AAV delivery to achieve knockout at multiple genomic sites.

FIG. 8A - FIG. 8D show data related to implementation of Cas9 mRNA/AAV guide delivery to generate CRISPR-mediated double knockout in primary human T-cells with E40RF6/E1B55K H373A expression.

FIG. 9 shows data related to implementation of Cas9 mRNA/AAV guide delivery to generate CRISPR-mediated knockout of more than two genes in primary human T-cells with E40RF6/E1B55K H373A expression.

FIG. 10A - FIG. 10C show data related the effect of using E1B55K mutants (E40RF6/E1B55K) enhance targeted CRISPR knock-in.

FIG. 11 shows data related to the effect of addition of E40RF6/E1B55K H373A or H354 on homology directed repair (HDR) when using shorter homology arms for gene knock-in with CRISPR-Cas9 at the CCR5 locus.

FIG. 12A - FIG. 12C show data related to the effect of addition of E40RF6/E1B55K H354 on HDR when using shorter homology arms for gene knock-in with CRISPR-Cas9 at the TCR locus.

FIG. 13A - FIG. 13B show data related to surface marker phenotype and Ca2+ signaling in edited primary T-cells with E40RF6/E1B55K H373A.

FIG. 14A - FIG. 14B show data related to cell karyotype following multiplex CRISPR editing.

FIG. 15A - FIG. 15C show data related to molecular confirmation of HDR events following CRISPR-Cas9 breaks with E40RF6/E1B55K H373A or H354.

FIG. 16A shows alternatives of polynucleotide sequences of guide RNAs guide1 (SEQ ID NO: 15), guide2 (SEQ ID NO: 16), guide3 (SEQ ID NO: 17), and guide4 (SEQ ID NO: 5) used for generating TCR knockout using the CRISPR/Cas9 system.

FIG. 16B shows flow cytometry data comparing the efficiency of Cas9-mCherry expression from mRNA in primary T-cells from donor 1 when using guide RNAs guide1-guide4.

FIG. 16C shows flow cytometry data comparing the efficiency of generation of TCR knockout in primary T-cells from donor 1 when using guide RNAs guide1-guide4.

<para-num num=

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 15/152,960 filed on May 12, 2016 which claims the benefit of U.S. Prov. App. No. 62/161,104 filed on May 13, 2015, which are each hereby expressly incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled SCRI094D1SEQLIST created on Jan. 10, 2020 and which is approximately 70 kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

Aspects of the disclosure provided herein are generally related to endonuclease-based gene editing systems and methods. Some aspects of the disclosure provided herein are related to the CRISPR/Cas9 gene editing system.

BACKGROUND

Endonuclease-based systems have rapidly become significant gene editing tools in biomedical research, with their application for gene disruption and/or gene targeting demonstrated in a variety of cultured cell and model organism systems.

Endonuclease-based systems for gene editing allow scientists to edit genomes with unprecedented precision, efficiency, and flexibility. Examples of endonuclease-based approaches for gene editing include systems comprising, without limitations, zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases (such as MegaTALs), and CRISPR/Cas9.

SUMMARY

The present disclosure provides several methods for applying CRISPR/Cas9 in primary cells in which an mRNA is used to express Cas9, and simultaneously, mRNA is used to transiently express two adenoviral proteins, E40RF6 and an H373A or H354 mutant version of E1B55K. The wild type E40RF6 and EB55K proteins relieve post-entry defects for expression from AAV vectors; however, if the wild type EB55K or E40RF6 proteins are used, they disable an important protein complex involved in DNA repair (known as the MRN complex), which leads to cell cycle arrest and high toxicity due to lack of repair of DNA breaks. Instead of using the wild type proteins mutants of E1B55K, which do not disable the MRN complex are utilized. Co-expression of Cas9 with E40RF6/E1B55K mutants results in sufficient relief of the post-entry restriction on AAV expression while maintaining intact DNA repair. This allows for a substantial improvement in Cas9-mediated gene editing efficiency with minimal toxicity when an AAV vector is used to express the guide RNA&#39;s necessary for Cas9 targeting.

Some alternatives of the system provided herein, comprise endonucleases so as to provide additional tools useful in gene disruption. Several alternatives, for example, relate to systems utilizing CRISPR/Cas9 systems and methods for enhancing the efficiency of inactivation of a target gene concurrently with endonucleases. More alternatives relate to the inactivation of a target gene for therapeutic, agricultural and/or other commercially useful purposes utilizing one or more of the systems described herein. Still more alternatives relate to the production of autologous and/or non-autologous primary cells having an inactivated target gene and the use of these cells for therapeutic and/or other commercial applications.

In some alternatives, a system for editing at least one target gene in a cell is provided, the system comprising a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector; a second nucleic acid sequence encoding a Cas9 protein, a derivative or fragment thereof; a third nucleic acid sequence encoding a first adenoviral protein; and a fourth nucleic acid sequence encoding a second adenoviral protein. In some alternatives of the system, the cell is a eukaryotic cell. In some alternatives of the system, the cell is a mammalian cell. In some alternatives of the system, the cell is a human cell. In some alternatives of the system, the cell is a primary cell. In some alternatives of the system, the cell is not a transformed cell. In some alternatives of the system, the cell is a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell or an intestinal cell.

In some alternatives of the system, the vector is a viral vector. In some alternatives of the system, the viral vector is an Adeno-associated virus (AAV) vector. In some alternatives of the system, the second nucleic acid encoding the Cas9 protein, a derivative or fragment thereof is an mRNA. In some alternatives of the system, the second nucleic acid sequence encoding the Cas9 protein is codon optimized for expression in a eukaryotic cell, such as a human cell. In some alternatives of the system, the Cas9 protein, a derivative or fragment thereof is from S. pyogenes . In some alternatives of the system, the third nucleic acid encoding the first adenoviral protein is an mRNA. In some alternatives of the system, the mRNA is codon optimized for expression in a eukaryotic cell, such as a human cell. In some alternatives of the system, the first adenoviral protein is E40RF6. In some alternatives of the system, the fourth nucleic acid encoding the second adenoviral protein is an mRNA. In some alternatives of the system, the fourth nucleic acid encoding the second adenoviral protein is codon optimized for expression in a eukaryotic cell, such as a human cell. In some alternatives of the system, the second adenoviral protein is an E1B55K mutant. In some alternatives of the system, the first, second, third and fourth nucleic acid sequences are joined to regulatory elements that are operable in a eukaryotic cell, such as a human cell. In some alternatives of the system, the first nucleic acid sequence encoding the CRISPR guide RNA is operably linked to a regulatory element. In some alternatives of the system, the nucleic acid sequence encoding the CRISPR guide RNA is operably linked to a promoter, for example, a U6 promoter. In some alternatives of the system, the first nucleic acid sequence encoding the CRISPR guide RNA is constitutively expressed.

In some alternatives, a method of editing at least one target gene in a cell is provided, the method comprising introducing into a cell a first vector that comprises a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in said cell; introducing into said cell a second nucleic acid sequence encoding a Cas9 protein, a derivative or fragment thereof, introducing into said cell a third nucleic acid sequence encoding a first adenoviral protein; and introducing into said cell a fourth nucleic acid sequence encoding a second adenoviral protein. In some alternatives of the method, the cell is a eukaroytic cell. In some alternatives of the method, the cell is a mammalian cell. In some alternatives of the method, the cell is a human cell. In some alternatives of the method, the cell is a primary cell. In some alternatives of the method, the cell is not a transformed cell. In some alternatives of the method, the cell is a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell or an intestinal cell.

In some alternatives of the method, the first vector comprising the first nucleic acid sequence encoding the CRISPR guide RNA is a viral vector. In some alternatives of the method, the viral vector is an Adeno-associated virus (AAV) vector. In some alternatives of the method, the second, third and fourth nucleic acid sequences are mRNA. In some alternatives of the method, the mRNAs are codon optimized for expression in a eukaryotic cell, such as a human. In some alternatives of the method, the Cas9 protein, a derivative or fragment thereof is from S. pyogenes . In some alternatives of the method, the first adenoviral protein is E40RF6. In some alternatives of the method, the second adenoviral protein is a EB55K mutant. In some alternatives of the method, the CRISPR guide RNA is complimentary to a target gene of interest. In some alternatives of the method, the CRISPR guide RNA is complimentary to a target gene of interest. In some alternatives of the method, the first, second, third and fourth nucleic acid sequences are transiently introduced into the cell. In some alternatives of the method, the first, second, third and fourth nucleic acid sequences are not permanently introduced into the cell. In some alternatives of the method, the introducing of the first, second, third and fourth nucleic acid sequences into the cell does not transform the cell. In some alternatives of the method, the target gene is a selected or identified gene of interest. In some alternatives of the method and/or the system, the second, third, or fourth nucleic acid sequence is provided on a vector. In some alternatives, a method of editing at least one target gene in a cell is practiced by introducing into a cell any of the alternatives of the system described herein.

In some alternatives, a method of treating, ameliorating, or inhibiting a disease and/or a condition in a subject is practiced by providing to a subject having a disease and/or a condition any of the alternatives of the system described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of mRNA and AAV vector constructs used for TCR locus targeting.

FIG. 1B shows T7 assay of editing-induced insertions-deletions (indels) at TCRα locus.

FIG. 1C shows the effect of Cas9-T2A-mCherry mRNA dose on expression at 24 h post transfection.

FIG. 1D shows the effect of increasing AAV dose on TCRα knockout.

FIG. 1E shows flow cytometry data related to comparison of TCRα knockout with single stranded (ss) versus self-complementary (sc) AAV for guide expression in T-cells.

FIG. 2A shows data related to relief of post-entry restriction of AAV-mediated gene expression.

FIG. 2B shows data related to comparison of effect of E40RF6/E1B55K proteins on self-complementary and single-stranded AAV6-mediated gene expression

FIG. 2C shows data related to MRN inactivation and its effect of relief of post-entry restriction on AAV-mediated expression.

FIG. 3A shows the effect of E40RF6/E1B55K mutants on AAV-driven GFP expression.

FIG. 3B shows the effect of E40RF6/E1B55K-H373 expression on AAV transduction.

FIG. 3C shows a comparison of E1B55K, E40RF6 mutants effects on AAV transduction.

FIG. 3D shows a comparison of E1B55K, E40RF6 mutants effects on AAV-driven GFP expression.

FIG. 4A - FIG. 4C show data related to CRISPR-mediated gene knockout in primary human T-cells through use of adenoviral E40RF6/E1B55K proteins.

FIG. 5A - FIG. 5C show data related to the effect of E40RF6/E1B55K MRN mutants on indel spectra.

FIG. 6 shows the effect of E40RF6/E1B55K proteins on non-homologous AAV insertion.

FIG. 7A - FIG. 7B show data related to implementation of CRISPR/Cas9 with mRNA/AAV delivery to achieve knockout at multiple genomic sites.

FIG. 8A - FIG. 8D show data related to implementation of Cas9 mRNA/AAV guide delivery to generate CRISPR-mediated double knockout in primary human T-cells with E40RF6/E1B55K H373A expression.

FIG. 9 shows data related to implementation of Cas9 mRNA/AAV guide delivery to generate CRISPR-mediated knockout of more than two genes in primary human T-cells with E40RF6/E1B55K H373A expression.

FIG. 10A - FIG. 10C show data related the effect of using E1B55K mutants (E40RF6/E1B55K) enhance targeted CRISPR knock-in.

FIG. 11 shows data related to the effect of addition of E40RF6/E1B55K H373A or H354 on homology directed repair (HDR) when using shorter homology arms for gene knock-in with CRISPR-Cas9 at the CCR5 locus.

FIG. 12A - FIG. 12C show data related to the effect of addition of E40RF6/E1B55K H354 on HDR when using shorter homology arms for gene knock-in with CRISPR-Cas9 at the TCR locus.

FIG. 13A - FIG. 13B show data related to surface marker phenotype and Ca2+ signaling in edited primary T-cells with E40RF6/E1B55K H373A.

FIG. 14A - FIG. 14B show data related to cell karyotype following multiplex CRISPR editing.

FIG. 15A - FIG. 15C show data related to molecular confirmation of HDR events following CRISPR-Cas9 breaks with E40RF6/E1B55K H373A or H354.

FIG. 16A shows alternatives of polynucleotide sequences of guide RNAs guide1 (SEQ ID NO: 15), guide2 (SEQ ID NO: 16), guide3 (SEQ ID NO: 17), and guide4 (SEQ ID NO: 5) used for generating TCR knockout using the CRISPR/Cas9 system.

FIG. 16B shows flow cytometry data comparing the efficiency of Cas9-mCherry expression from mRNA in primary T-cells from donor 1 when using guide RNAs guide1-guide4.

FIG. 16C shows flow cytometry data comparing the efficiency of generation of TCR knockout in primary T-cells from donor 1 when using guide RNAs guide1-guide4.

FIG. 16D shows flow cytometry data comparing Cas9-mCherry expression levels in primary T-cells from donor 1 when using different volumes of Cas9/guide sample.

FIG. 16E shows flow cytometry data comparing the efficiency of generation of TCR knockout in primary T-cells from donor 1 when using different volumes of sample containing guide RNA guide4.

FIG. 16F shows flow cytometry data comparing Cas9-mCherry expression levels in primary T-cells from donor 2 when using different volumes of Cas9/guide sample containing guide RNA guide4.

FIG. 16G shows flow cytometry data comparing the efficiency of generation of TCR knockout in primary T-cells from donor 2 when using different volumes of sample containing guide RNA guide4.

FIG. 16H shows flow cytometry data comparing the efficiency of Cas9-mCherry expression from mRNA in Jurkat T-cells when using guide RNAs guide1-guide4.

FIG. 16I shows flow cytometry data comparing the efficiency of generation of TCR knockout in Jurkat T-cells when using guide RNAs guide1-guide4.

FIG. 17 shows the protein sequence of an alternative of a wild type adenoviral protein E1B55K (SEQ ID NO: 1).

FIG. 18 shows the protein sequence of an alternative of a mutant adenoviral protein E1B55K with an H373A polymorphism (SEQ ID NO: 2). The mutation is shown in bold and underlined.

FIG. 19 shows the protein sequence of an alternative of a wild type adenoviral protein E40RF6 (SEQ ID NO: 3).

FIG. 20 shows the protein sequence of an alternative of a mutant adenoviral protein E1B55K with an H354 (SEQ ID NO: 4). The mutation/insertion is shown in bold and underlined.

FIG. 21 shows flow cytometry data of generation of TCRα knockout with CRISPR guide RNAs guide1 (G1), guide2 (G2), guide3 (G3) and guide4 (G4).

FIG. 22 shows the protein sequence of an alternative dCas9 variant of Cas9 protein of Streptococcus pyogenes (SEQ ID NO: 6).

FIG. 23 shows the nucleotide sequence of an alternative Cas9-SP variant from Streptococcus pyogenes (SEQ ID NO: 7).

FIG. 24 shows the nucleotide sequence of an alternative Cas9-SPm4 variant from Streptococcus pyogenes (SEQ ID NO: 8).

FIG. 25 shows the nucleotide sequence of an alternative Cas9-ST1 variant from Streptococcus thermophilus (SEQ ID NO: 9).

FIG. 26 shows the nucleotide sequence of an alternative Cas9-ST1m4 variant from Streptococcus thermophilus (SEQ ID NO: 10).

FIG. 27 shows the nucleotide sequence of an alternative Cas9-NM variant from Neisseria meningitidis (SEQ ID NO: 11).

FIG. 28 shows the nucleotide sequence of an alternative Cas9-NMm4 variant from Neisseria meningitidis (SEQ ID NO: 12).

FIG. 29 shows the nucleotide sequence of an alternative Cas9-TD variant from Treponema denticola (SEQ ID NO: 13).

FIG. 30 shows the nucleotide sequence of an alternative Cas9-TDm4 variant from Treponema denticola (SEQ ID NO: 14).

FIG. 31 shows alternatives of polynucleotide sequences of PD1 guide RNA (SEQ ID NO: 18), TIGIT guide RNA (SEQ ID NO: 19), Lag3 guide RNA (SEQ ID NO: 20) and Tim3 guide RNA (SEQ ID NO: 21).

FIG. 32A shows the effect of TALENs on HDR for the CCR5 locus.

FIG. 32B shows the effect of TALENs on HDR for the CD40L locus.

FIG. 33 shows the nucleotide sequence of an alternative of an R240A mutant of the adenoviral protein E1B55K (SEQ ID NO: 22).

FIG. 34 shows the nucleotide sequence of an alternative of an AXA mutant of the adenoviral protein E40RF6 (SEQ ID NO: 23).

DETAILED DESCRIPTION

In some alternatives, nuclease-based gene editing systems and methods are provided. Examples of nuclease-based approaches for gene editing include systems comprising nucleases such as, without limitations, ZFNs, TALENs, meganucleases (e.g., MegaTALs) and CRISPR/Cas9.

The gene-editing systems and methods provided herein can be applied to any nuclease-based gene editing approach comprising, without limitations, gene disruption and/or gene targeting. For example, aspects of the present disclosure are related to CRISPR/Cas9-based gene editing. In some alternatives, Cas9 nuclease-mediated enhancement of gene editing is provided.

An important aspect of applying CRISPR/Cas9 for gene editing is the need for a system to express the guide RNA&#39;s efficiently in a wide variety of cell types. An important system for expressing guide RNAs is based on the use of adeno-associated virus vectors (AAV). AAV vectors are able to transduce a wide range of primary cells.

However, in many cell types, there is a post-entry restriction on AAV vectors that renders AAV-mediated expression of transgenes, including guide RNAs, very inefficient, thus substantially compromising the utility of AAV vectors for this purpose. Therefore, an approach to substantially improve and expand the potential applications of the CRISPR/Cas9 system in primary cells is contemplated.

In some alternatives, Cas9-based approach enhances gene editing efficiency with minimal toxicity when adeno-associated virus vectors (AAV) are used to express the guide RNA&#39;s necessary for Cas9 targeting.

CRISPR/Cas9 and related programmable endonuclease systems have rapidly become significant gene editing tools of the biomedical research laboratory, with their application for gene disruption and/or gene targeting demonstrated in a variety of cultured cell and model organism systems. Although the flexibility with which the Cas9 nuclease can be re-programmed to target new sites is a major advantage for genome engineering in the research setting, several practical barriers limit the direct extension of research-based gene editing methods to editing of primary human cells for therapeutic purposes.

Examples of some of these practical barriers include limited opportunities to identify and enrich for cells that have incurred a desired editing event; the requirement for transient (e.g. a few days) nuclease delivery due to safety and immunogenicity issues associated with longer term and/or in vivo nuclease expression; and limitations in vector systems for nuclease or recombination template delivery posed by primary cells&#39; robust capacity to detect the presence of cytosolic DNA and consequent generation of anti-viral or pro-apoptotic signals.

Driven by the practical barriers delineated herein, therapeutic gene editing strategies utilizing zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and meganucleases, have gravitated towards delivery approaches that ensure transient nuclease expression, most notably mRNA transfection, and the use of viral vectors for recombination template delivery. For these same reasons, mRNA-based CRISPR component expression has recently been extended to human primary cells for the purpose of gene disruption through the use of electroporation to deliver Cas9 mRNA or protein in conjunction with either native or degradation-resistant guide RNAs.

While RNA or protein/RNA-based nuclease delivery are straightforward methods for disrupting individual genes, applications of CRISPR-based gene editing that involve gene targeting require efficient delivery of three components: Cas9, guide RNA, and a recombination template.

In some alternatives, an electroporation/transduction co-delivery method for CRISPR/Cas9 gene editing that utilizes mRNA electroporation-mediated expression of Cas9 in conjunction with variants of two adenoviral serotype 5 proteins, E40RF6 and E1B55K is provided, that transiently enhance both primary cells&#39; capacity for transduction by AAV and gene editing efficiency.

In some alternatives, using a cell culture/manufacturing protocol compatible with clinical translation, the application of this method for efficient gene disruption and homology-directed gene targeting in primary human T-cells is provided.

Definitions

In the description that follows, a number of terms are used extensively. The following definitions are provided to facilitate understanding of the present alternatives.

As used herein, “a” or “an” may mean one or more than one.

As used herein, the term “about” indicates that a value includes the inherent variation of error for the method being employed to determine a value, or the variation that exists among experiments.

As used herein, “nucleic acid” or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. The term “nucleic acid molecule” also includes so-called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded. The basic components of CRISPR/Cas9 system comprise a target gene, a guide RNA, and a Cas9 endonuclease, derivative, or fragment thereof. An important aspect of applying CRISPR/Cas9 for gene editing is the need for a system to deliver the guide RNAs efficiently to a wide variety of cell types. This could for example involve delivery of an in vitro generated guide RNA as a nucleic acid (the guide RNA generated by in vitro transcription or chemical synthesis). In some alternatives the nucleic acid encoding the guide RNA is rendered nuclease resistant by incorporation of modified bases, such as 2′O-methyl bases. In some alternatives, the CRISPR/Cas9 system described herein, whereby the polynucleotide encoding the Cas9 nuclease or a derivative or functional fragment thereof (e.g., SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14) is provided with a poly(T) or poly(A) tail of a desired length and prepared in accordance with the teachings described herein, for example, is provided with a guide RNA that comprises one or more modified bases, such as any one or more of the modified bases described herein.

Exemplary guide RNAs useful with the alternatives described herein, which may contain one or more of the modified bases set forth herein are provided in SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. Furthermore, an important system for expressing guide RNAs in this context is based on the use of adeno-associated virus (AAV) vectors because AAV vectors are able to transduce a wide range of primary cells. AAV vectors do not cause infection and are not known to integrate into the genome. Therefore, the use of AAV vectors has the benefits of being both safe and efficacious.

The term “complementary to” means that the complementary sequence is homologous to all or one or more portions of a reference polynucleotide sequence. For illustration, the nucleotide sequence “CATTAG” corresponds to a reference sequence “CATTAG” and is complementary to a reference sequence “GTAATC.”

A “promoter” is a nucleotide sequence that directs the transcription of a structural gene. In some alternatives, a promoter is located in the 5′ non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993); incorporated by reference in its entirety herein), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol. 1:47 (1990); incorporated by reference in its entirety herein), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE/ATF (O&#39;Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994)), SPi, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1 (1994); all references incorporated by reference in their entireties herein). As used herein, a promoter may be constitutively active, repressible or inducible. If a promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known. In some alternatives, a regulatory element can be an untranslated region. In some alternatives, an untranslated region is a 5′ untranslated region. In some alternatives, an untranslated region is a 3′ untranslated region. In some alternatives, either 5′ or 3′ untranslated region is used. In some alternatives, both 5′ and 3′ untranslated regions are used. One skilled in the art will understand the meaning of an untranslated region as used in the alternatives here.

A “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter. For example, a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a “cell-specific,” “tissue-specific,” or “organelle-specific” manner. In some alternatives, a system for editing at least one target gene in a cell is provided, wherein the system comprises a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector; said system also comprising a second nucleic acid sequence encoding a Cas9 protein, a third nucleic acid sequence encoding a first adenoviral protein, and a fourth nucleic acid sequence encoding a second adenoviral protein. In some alternatives, the first, second, third and fourth nucleic acid sequences are joined to regulatory elements that are operable in a eukaryotic cell, such as a human cell.

A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.” A polypeptide can be considered as a protein.

A “protein” is a macromolecule comprising one or more polypeptide chains. A protein may also comprise non-peptide components, such as carbohydrate groups. Carbohydrates and other non-peptide substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless. In some embodiments, a system for editing at least one target gene in a cell is provided, wherein the method comprises a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector; said system also comprising a second nucleic acid sequence encoding a Cas9 protein, a third nucleic acid sequence encoding a first adenoviral protein and a fourth nucleic acid sequence encoding a second adenoviral protein.

As used herein, “transient transfection” refers to the introduction of exogenous nucleic acid(s) into a host cell by a method that does not generally result in the integration of the exogenous nucleic into the genome of the transiently transfected host cell. In some alternatives, the nucleic acid is RNA. In some alternatives, the nucleic acid is DNA. In some alternatives, when the nucleic acid is RNA, the nucleic acid does not generally integrate in the genome of the transiently transfected cell. In some alternatives, when the nucleic acid is DNA, the nucleic acid can integrate in the genome of the transiently transfected cell.

By the term “host cell” is meant a cell that is introduced with Cas9-mRNA/AAV-guide RNA according to the present alternatives, as well as, cells that are provided with the systems herein. Host cells can be prokaryotic cells or eukaryotic cells. Examples of prokaryotic host cells include, but are not limited to E. coli , nitrogen fixing bacteria, Staphylococcus aureus, Staphylococcus albus, Lactobacillus acidophilus, Bacillus anthracis, Bacillus subtilis, Bacillus thuringiensis, Clostridium tetani, Clostridium botulinum, Streptococcus mutans, Streptococcus pneumoniae , mycoplasmas, and cyanobacteria. Examples of eukaryotic host cells include, but are not limited to, protozoa, fungi, algae, plant, insect, amphibian, avian and mammalian cells. In some alternatives, a system for editing at least one target gene in a cell is provided, wherein the cell is a eukaryotic cell. In some alternatives, the cell is a mammalian cell. In some alternatives, the cell is a human cell. In some alternatives, the cell is a primary cell. In some alternatives, the cell is not a transformed cell. In some alternatives, the cell is a primary lymphocyte. In some alternatives, the cell is a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell or an intestinal cell.

The term “gene expression” refers to the biosynthesis of a gene product. For example, in the case of a structural gene, gene expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.

The term “endonuclease” refers to enzymes that cleave the phosphodiester bond within a polynucleotide chain. The polynucleotide may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (for example, containing bases other than A, C, G, and T). An endonuclease may cut a polynucleotide symmetrically, leaving “blunt” ends, or in positions that are not directly opposing, creating overhangs, which may be referred to as “sticky ends.” The methods and compositions described herein may be applied to cleavage sites generated by endonucleases. In some alternatives of the system, the system can further provide nucleic acids that encode an endonuclease, such as Cas9, TALEN, or MegaTAL, or a fusion protein comprising a domain of an endonuclease, for example, Cas9, TALEN, or MegaTAL, or one or more portion thereof. These examples are not meant to be limiting and other endonucleases and alternatives of the system and methods comprising other endonucleases and variants and modifications of these exemplary alternatives are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

The term “TAL Effector Nuclease” (TALEN) refers to a nuclease comprising a TAL-effector domain fused to a nuclease domain. TAL-effector DNA binding domains, isolated from the plant pathogen Xanthomonas have been described (see Boch et al., (2009) Science 29 Oct. 2009 (10.1126/science.117881) and Moscou and Bogdanove, (2009) Science 29 Oct. 2009 (10.1126/science.1178817); both references incorporated by reference in their entireties herein). These DNA binding domains may be engineered to bind to a desired target and fused to a nuclease domain, such as the Fok1 nuclease domain, to derive a TAL effector domain-nuclease fusion protein. The methods and systems described herein may be applied to cleavage sites generated by TAL effector nucleases. In some alternatives of the systems provided herein, the systems can further comprise a TALEN nuclease or a vector or nucleic acid encoding a TALEN nuclease. In some alternatives of the methods provided herein, the method can further comprise providing a nuclease, such as a TALEN nuclease.

MegaTALs are derived from the combination of two distinct classes of DNA targeting enzymes. Meganucleases (also referred to as homing endonucleases) are single peptide chains that have the advantage of both DNA recognition and nuclease functions in the same domain. In some alternatives of the systems provided herein, the systems can further comprise a MegaTAL nuclease or a vector or nucleic acid encoding a MegaTAL nuclease. In some alternatives of the methods provided herein, the methods can further comprise providing MegaTAL nuclease or a vector or nucleic acid encoding a MegaTAL nuclease.

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes , among other bacteria. S. pyogenes utilizes Cas9 to memorize and later interrogate and cleave foreign DNA, such as invading bacteriophage DNA or plasmid DNA. Cas9 performs this interrogation by unwinding foreign DNA and checking for if it is complementary to the 20 base pair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA.

CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a bacterial virus or plasmid. CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life. By delivering the Cas9 protein, a derivative, or fragment thereof and appropriate guide RNAs into a cell, the organism&#39;s genome can be cut at any desired location. It can be possible to use CRISPR to build RNA-guided gene drives capable of altering the genomes of entire populations. In some alternatives, a system for editing at least one target gene in a cell is provided, wherein the method comprises a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector, a second nucleic acid sequence encoding a Cas9 protein, a derivative, or fragment thereof, a third nucleic acid sequence encoding a first adenoviral protein and a fourth nucleic acid sequence encoding a second adenoviral protein. Exemplary guide RNAs useful with the alternatives described herein, which may contain one or more modified bases, are provided in SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and/or SEQ ID NO: 21.

In some alternatives, the use of chemically modified guide RNAs is contemplated. Chemically-modified guide RNAs have been used in CRISPR-Cas genome editing in human primary cells (Hendel, A. et al., Nat Biotechnol. 2015 September; 33(9):985-9), expressly incorporated by reference. Chemical modifications of guide RNAs can include modifications that confer nuclease resistance. Nucleases can be endonucleases, or exonucleases, or both. Some chemical modification, without limitations, include 2′-fluoro, 2′O-methyl, phosphorothioate dithiol 3′-3′ end linkage, 2-amino-dA, 5-mehtyl-dC, C-5 propynyl-C, C-5 propynyl-U, morpholino, etc. These examples are not meant to be limiting and other chemical modifications and variants and modifications of these exemplary alternatives are also contemplated.

The term “exonuclease” refers to enzymes that cleave phosphodiester bonds at the end of a polynucleotide chain via a hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or 5′ end. The polynucleotide may be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (for example, containing bases other than A, C, G, and T). The term “5′ exonuclease” refers to exonucleases that cleave the phosphodiester bond at the 5′ end. The term “3′ exonuclease” refers to exonucleases that cleave the phosphodiester bond at the 3′ end. Exonucleases may cleave the phosphodiester bonds at the end of a polynucleotide chain at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (for example by passing through fine-gauge needle, heating, sonicating, mini bead tumbling, and nebulizing), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolosis and chemotherapy agents. Exonucleases may cleave the phosphodiester bonds at blunt ends or sticky ends. E. coli exonuclease I and exonuclease III are two commonly used 3′-exonucleases that have 3′-exonucleolytic single-strand degradation activity. Other examples of 3′-exonucleases include Nucleoside diphosphate kinases (NDKs), NDK1 (NM23-H1), NDK5, NDK7, and NDK8 (Yoon J-H, et al., Characterization of the 3′ to 5′ exonuclease activity found in human nucleoside diphosphate kinase 1 (NDK1) and several of its homologues. (Biochemistry 2005:44(48):15774-15786.), WRN (Ahn, B., et al., Regulation of WRN helicase activity in human base excision repair. J. Biol. Chem. 2004, 279:53465-53474) and Three prime repair exonuclease 2 (Trex2) (Mazur, D. J., Perrino, F. W., Excision of 3′ termini by the Trex1 and TREX2 3′→5′ exonucleases. Characterization of the recombinant proteins. J. Biol. Chem. 2001, 276:17022-17029; both references incorporated by reference in their entireties herein). E. coli exonuclease VII and T7- exonuclease Gene 6 are two commonly used 5′-3′ exonucleases that have 5% exonucleolytic single-strand degradation activity. The exonuclease can be originated from prokaryotes, such as E. coli exonucleases, or eukaryotes, such as yeast, worm, murine, or human exonucleases. In some alternatives of the systems provided herein, the systems can further comprise an exonuclease or a vector or nucleic acid encoding an exonuclease. In some alternatives, the exonuclease is Trex2. In some alternatives of the methods provided herein, the methods can further comprise providing exonuclease or a vector or nucleic acid encoding an exonuclease, such as Trex2.

The term “cleavage” refers to the breakage of the covalent backbone of a polynucleotide. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. Double stranded DNA, RNA, or DNA/RNA hybrid cleavage can result in the production of either blunt ends or staggered ends.

“Prokaryotic” cells lack a true nuclease. Examples of prokaryotic cells are bacteria (e.g., cyanobacteria, Lactobacillus acidophilus , Nitrogen-Fixing Bacteria, Helicobacter pylori, Bifidobacterium, Staphylococcus aureus, Bacillus anthrax, Clostridium tetani, Streptococcus pyogenes, Staphylococcus pneumoniae, Klebsiella pneumoniae and Escherichia coli ) and archaea (e.g., Crenarchaeota, Euryarchaeota, and Korarchaeota). The Cas9 protein described herein is a protein from a prokaryotic cell.

“Eukaryotic” cells include, but are not limited to, algae cells, fungal cells (such as yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T-cells).

The term “subject” as used herein includes all members of the animal kingdom including non-human primates and humans. In some alternatives, a system for editing at least one target gene in a cell is provided, wherein the method comprises a first nucleic acid sequence encoding a CRISPR guide RNA, wherein the CRISPR guide RNA is complimentary to at least one target gene in a cell and, wherein said first nucleic acid sequence is present in a vector, a second nucleic acid sequence encoding a Cas9 protein, a derivative, or fragment thereof, a third nucleic acid sequence encoding a first adenoviral protein and a fourth nucleic acid sequence encoding a second adenoviral protein. In some alternatives, the cell that comprises an edited gene is delivered to a subject in need.

The homing endonucleases, also known as meganucleases, are sequence specific endonucleases that generate double strand breaks in genomic DNA with a high degree of specificity due to their large (e.g., &gt;14 bp) cleavage sites. While the specificity of the homing endonucleases for their target sites allows for precise targeting of the induced DNA breaks, homing endonuclease cleavage sites are rare and the probability of finding a naturally occurring cleavage site in a targeted gene is low. In some alternatives of the systems provided herein, the systems can further comprise a meganuclease or a vector or nucleic acid encoding a meganuclease. In some alternatives of the methods provided herein, the methods can further comprise providing a meganuclease or a vector or nucleic acid encoding a meganuclease.

Another class of artificial endonucleases is the engineered meganucleases. Engineered homing endonucleases are generated by modifying the specificity of existing homing endonucleases. In one approach, variations are introduced in the amino acid sequence of naturally occurring homing endonucleases and then the resultant engineered homing endonucleases are screened to select functional proteins which cleave a targeted binding site. In another approach, chimeric homing endonucleases are engineered by combining the recognition sites of two different homing endonucleases to create a new recognition site composed of a half-site of each homing endonuclease. In some alternatives of the systems provided herein, the systems can further comprise an engineered meganuclease or a vector or nucleic acid encoding an engineered meganuclease.

<div id="p-0103" num="0102" class="descri

CLAIMS

Claims ( 21 )

1 . A method for editing a target gene in a cell, the method comprising:

introducing into a cell a first nucleic acid wherein the first nucleic acid encodes a CRISPR guide RNA complimentary to the target gene; introducing into the cell a Cas9 protein or a second nucleic acid encoding a Cas9 protein; introducing into the cell a third nucleic acid encoding a first adenoviral protein; and introducing into the cell a fourth nucleic acid encoding a second adenoviral protein.

2 . The method of claim 1 , wherein the first adenoviral protein and the second adenoviral protein are from a serotype 5 adeno-associated virus (AAV).

3 . The method of claim 1 , wherein the first adenoviral protein comprises a wild type E40RF6 protein, or an AXA mutant E40RF6 protein.

4 . The method of claim 3 , wherein the first adenoviral protein comprises the AXA mutant E40RF6 protein, and wherein the AXA mutant E40RF6 protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO:23.

5 . The method of claim 1 , wherein the second adenoviral protein comprises a wild type E1B55K protein, or a mutant EB55K protein.

6 . The method of claim 5 , wherein the second adenoviral protein comprises the mutant E1B55K protein, and wherein the mutant E1B55K protein is selected from the group consisting of an H373A mutant E1B55K protein, an H354 mutant E1B55K protein, and an R240A mutant E1B55K protein.

7 . The method of claim 6 , wherein the mutant E1B55K protein is the H373A mutant E1B55K protein and the H373A mutant E1B55K protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO:02.

8 . The method of claim 6 , wherein the mutant E1B55K protein is the H354 mutant E1B55K protein and the H354 mutant E1B55K protein comprises an amino acid sequence having at least 95% identity to SEQ ID NO:04.

9 . The method of claim 1 , wherein the CRISPR guide RNA comprises the nucleotide sequence of any one of SEQ ID NO:05, and SEQ ID NOs:15-21.

10 . The method of claim 1 , wherein a vector comprises the first nucleic acid.

11 . The method of claim 10 , wherein the vector is an adeno-associated virus (AAV) vector.

12 . The method of claim 11 , wherein the AAV vector is a self-complementary vector, or a single stranded vector.

13 . The method of claim 1 , wherein the second nucleic acid, the third nucleic acid and the fourth nucleic acid comprise RNA.

14 . The method of claim 1 , wherein a vector comprises the third nucleic acid and fourth nucleic acid.

15 . The method of claim 1 , wherein the Cas9 protein is a S. pyogenes Cas9 protein.

16 . The method of claim 1 , wherein introducing the first nucleic acid, introducing the second nucleic acid, introducing nucleic acid the third nucleic acid, and introducing the fourth nucleic acid, each comprises transiently introducing the nucleic acid into the cell.

17 . The method of claim 1 , wherein the first nucleic acid, second nucleic acid and third nucleic are introduced into the cell in an order selected from:

(i) the second nucleic acid is introduced into the cell prior to a vector comprising the first nucleic acid, the second nucleic acid, and the third nucleic acid is introduced into the cell; (ii) a vector comprising the first nucleic acid, the second nucleic acid, and the third nucleic acid is introduced into the cell prior to introducing the second nucleic acid into the cell; and (iii) the second nucleic acid and a vector comprising the first nucleic acid, the second nucleic acid, and the third nucleic acid are simultaneously introduced into the cell.

18 . The method of claim 1 , wherein the cell is a mammalian cell.

19 . The method of claim 1 , wherein the cell is selected from the group consisting of a primary lymphocyte, a CD34+ stem cell, a hepatocyte, a cardiomyocyte, a neuron, a glial cell, a muscle cell, and an intestinal cell.

20 . A method of treating, ameliorating, and/or inhibiting a disorder in a subject, the method comprising:

editing a target gene in cell according to the method of claim 1 to obtain an edited cell; and administering the edited cell to the subject.

21 . The method of claim 20 , wherein the disorder is selected from the group consisting of cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer&#39;s disease, muscular dystrophy, neurodegenerative disease, vascular disease, cystic fibrosis, stroke, hyper IGE syndrome, hemophilia achondroplasia, pseudoachondroplasia, multiple epiphyseal dysplasias, chondrodysplasias, osteogenesis imperfecta, Marfan syndrome, polydactyly, hereditary motor sensory neuropathies I and II (Charcot-Marie-Tooth disease), myotonic dystrophy, and neurofibromatosis.

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